The Drosophila Suzukii Mating Rate Decrease for the Reproductive Interference of Drosophila Melanogaster

Background: Drosophila suzukii has been widely distributed all over the world since 2008, and it is a harmful pest causing great economic loss in many countries. Previous research has found that the presence of Drosophila melanogaster could reduce the emergence and egg laying of Drosophila suzukii. In order to gure out the potential mechanism of this phenomenon, we studied three potential factors including lifetime, larval interspecic competition, and reproductive interference. Results: The results show that the Drosophila suzukii offspring number was signicantly decreased when reared with Drosophila melanogaster. The lifetime and larval interspecic competition have no signicant effect on the Drosophila suzukii population. Surprisingly, Drosophila melanogaster can cause reproductive interference with male Drosophila suzukii, which leads to a signicant decline in the successful mating rate of the latter fruit y. Conclusions: The presence of Drosophila melanogaster causes the Drosophila suzukii population to decrease through the effect of reproductive interference, and the Drosophila suzukii successful mating rate is signicantly decreased for the existence of Drosophila melanogaster.


Background
Drosophila suzukii (Diptera: Drosophilidae), which is also called spotted-wing drosophila (SWD), is originally from Asia, and has become a severe invasive pest in South America and Europe since 2008 [1,2], causing huge economic losses in the fruit production industry [3]. Usually, Drosophila suzukii lay eggs within ripe and ripening fruit by using a sclerotized and serrated ovipositor that can penetrate and lead to physical damage of fruit skins [4,5]. According to the literature, D. suzukii can lay up to 600 eggs (around 400 eggs on average) during their life span, and the minimum development time from oviposition to adult under optimal temperature is 8 days [6]. As for environmental adaptability, D. suzukii has a wide range of tolerance to climatic conditions, with a reproduction temperature from 10 °C up to 32 °C [7]. In addition, the activity and development temperature range are usually between 20 to 25 °C [8]. This pest also has wide host range, and up to now, there have been more than 150 species reported as hosts for D. suzukii [9,10]. Due to this, it is almost impossible to nd an effective way to eliminate this pest in a short time, for different hosts have non-identical ripening times, and wild hosts as well as ornamental plants can serve as refuges, so that D. suzukii can persist through survival challenges such as the low temperature in winter [11].
Currently, Drosophila suzukii has successfully invaded many areas and countries. The use of ovipositors can cause physical damage to intact fruit, and the larvae can speed up the process of decay, which provides an ideal food source to other phytophagous insects, so competition is almost inevitable with other Drosophila species, such as Drosophila melanogaster. Usually, competition includes intraspeci c competition and interspeci c competition. The outcome of competition depends on the relative strength of interspeci c competition and intraspeci c competition. Both types of competition are a struggle for survival for every individual because of limited environmental resources [12]. To plant-eating insects, the importance of interspeci c competition to community structure has been discussed by many ecologists [13,14]. Interspeci c competition widely exists among insects [15,16]. The research by Denno analyzed 193 pairs of competitor species, and concluded that the interspeci c competition affects the abundance and distribution of plant-eating insects [17]. The interspeci c competition between two species is an interspeci c interaction that could lead one species population to be reduced as the other species increases, which has great effect on species coexistence, habitat partitioning, food resource partitioning, and species replacement [18].
According to the niche theory, closely related species overlap in resource needs, so the competition can be very strong when two species rst meet [19]. A previous study concluded that, in a lab environment, the presence of D. melanogaster can signi cantly reduce emergence and egg laying of D. suzukii through interspeci c competition [20]. This is because most male Drosophila can produce a pheromone called cisvaccenyl acetate (cVA) that used in courtship, aggression, and aggregation signaling, which has a disruptive effect on D. suzukii [20]. The number of successful matings is reduced when male D. suzukii encounter this pheromone. Similarly, in previous work, we found that when adults of D. melanogaster and D. suzukii were reared together for three generations, the whole population of D. suzukii would eventually die. Thus, in this study, we attempted to explore more alternative potential factors.
The current study shows that both D. suzukii and D. melanogaster have strong appetites for rotten fruit, and have identical feeding niches. For acquiring resource, two species might show competition, which might cost time, energy and increase risk for injury. When interspeci c competition of two ies occurs, more time and energy might be used for competition, resulting in decreased in egg laying, and individuals might suffer physical damage, affecting the longevity of adults. Decreases in both egg laying and adult longevity could be important factors leading to the whole population of D. suzukii would be nally dying. Therefore, in our experiments, we investigated the effect of interspeci c competition on the longevity and fecundity of the two ies. In addition to adult competition, larval-larval competition between D. melanogaster and D. suzukii was also studied. Besides, that, reproductive interference is also a kind of interspeci c sexual interaction that the female reproductive success of one species is reduced because of the interference by another species [21]. The frequency of conspeci c courtship and mating, fecundity of females, and fertility of eggs can be in uenced by reproductive interference, and time, energy, nutrients, or gametes can be wasted in heterospeci c sexual interactions, causing tness loss for the individuals involved [22]. Reproductive interference might be a potential factor resulting in the population reduction of D. suzukii, and would be examined in this study.

Results
The offspring number and longevity comparison of two ies in two rearing conditions The D. suzukii offspring numbers were signi cantly decreased when rearing with D. melanogaster (Fig. 1a). Under independent rearing condition, the offspring number of D. suzukii (117.37 ± 24.28) was signi cantly higher than in mixed rearing condition (40.47 ± 8.23, t 17 = 3.13, P = 0.006). The same result also found in D. melanogaster that the offspring number in independent condition (494.50 ± 36.92) was extremely higher than in mixed rearing condition (269.53 ± 47.05, t 18 = 3.76, P < 0.01).
As for longevity, two ies were affected differently under two conditions (Fig. 1b). Our result showed that the lifetime of D. suzukii in the independent condition (54 ± 2.70 days) had no apparent difference comparing with the mixed condition (61.6 ± 3.04 days, t 36 = -1.85, P = 0.07). However, as for D.

The larval interspeci c competition of two ies
We calculated the number of pupation and eclosion of two ies in larval interspeci c competition experiments ( Table 1). The results showed that for D. suzukii, eclosion had no signi cant difference between independent and mixed rearing conditions (χ 2 = 3.52, df = 1, P = 0.061), which was similar as the result of pupation (χ 2 = 3.14, df = 1, P = 0.077). As for D. melanogaster, the eclosion number still did not have much difference between independent and mixed rearing conditions (χ 2 = 0.515, df = 1, P = 0.473), the pupation number had little difference between the two rearing conditions (χ 2 = 0.002, df = 1, P = 0.905). Table 1 The pupation and eclosion number in independent and mixed rearing conditions The reproductive interference could reduce the successful mating rate of D. suzukii We found the existence of D. melanogaster could impact the normal courtship and mating behavior (see Additional le e-h). And the D. suzukii successful mating rate was also affected ( behaviors also had no signi cant difference (U = 16, P = 0.472) between the two rearing conditions, the mating times were not signi cantly different between the independent (1738.1 ± 159.92 s) and mixed (1412.5 ± 182.75 s, t 12 =-1.16, P = 0.268) conditions.
As for D. melanogaster, the courtship times were also not signi cantly different in independent (442.54 ± 96.26 s) and mixed (202.3 ± 51.81 s, t 21 = 2.02, P = 0.057) conditions. However, the number of courtship behaviors before mating in the mixed rearing condition were signi cantly fewer than in the independent rearing condition (U = 102.5, P = 0.022), and the mating times were not signi cantly different between independent (987.69 ± 60.93 s) and mixed (961.9 ± 64.92 s, t 12 = 0.287, P = 0.777) conditions.
Referring to misdirected courtship, in mixed rearing conditions, the D. suzukii had signi cantly more misdirected courtship behavior than D. melanogaster (U = 40, P = 0.0057). Because the male D. suzukii had 13 times heterospeci c mating attempts towards female D. melanogaster. Surprisingly, the male D. melanogaster did not have this behavior in our observation. In addition, real heterospeci c mating happened twice between male D. suzukii and female D. melanogaster in behavior observation experiment.

Discussion
Similar to a previous study [20], when rearing mixed D. suzukii and D. melanogaster, our results showed that the offspring of D. suzukii signi cantly decreased (Fig. 1). Datta et al. (2008) suggested that existence of D. melanogaster could change the oviposition preference, resulting in reduced egg laying by D. suzukii. The male D. melanogaster may could produce a pheromone, cis-vaccenyl acetate (cVA) [23], which is natural repellent to female D. suzukii searching for oviposition sites. Our results suggested that, besides the oviposition preference change, reproductive interference by D. melanogaster was likely to be alternative factor resulting in the offspring of D. suzukii signi cantly decreasing.
Reproductive interference between invasive and native species has received much attention, and is a kind of interspeci c sexual interaction where the female reproductive success of one species is reduced because of the interference by another species [21]. In our behavior experiment, we found that the presence of female and male D. melanogaster had reproductive interference towards male D. suzukii, in which male D. suzukii might choose D. melanogaster female as the mating target (see Additional le f). Usually, reproductive interference is distinguished by seven types: signal jamming, heterospeci c rivalry, misdirected courtship, heterospeci c mating attempts, erroneous female choice, heterospeci c mating and hybridization [24]. Three types of this reproductive interference, including misdirected courtship, heterospeci c mating attempts and heterospeci c mating, were observed in our study.
In tested adults, 95% (19 of 20 groups) of D. suzukii males showed misdirected courtship behavior. Usually, misdirected courtship is performed or initiated by males, which are often indiscriminate in mate choice, as they invest less energy in reproduction [25], in our study, we found that misdirected courtship was initiated by male D. suzukii as well. These males may fail to recognize conspeci c females and may prefer heterospeci c females or even males [26,27]. The previous study reported that heterospeci c females with larger body size had more attraction to males because a large body size indicates high fecundity [28,29], which is contrary to our observations where male D. suzukii were attracted by female D. melanogaster that are smaller than female D. suzukii. Therefore, we deduce that male D. suzukii fail to recognize conspeci c females, and female D. melanogaster possess other features that can attract male D. suzukii. A study has shown that D. melanogaster sex pheromones are cuticular hydrocarbons (CHC) that mediate chemical communication for both sex and species recognition [30], which may make female D. melanogaster more attractive, and this CHC may prevent conspeci c female scent attraction for male D. suzukii.
Consistent with previous reports [31], in our observation experiments, all of heterospeci c mating attempts followed the misdirected courtship, while D. melanogaster males showed no heterospeci c mating attempts. The behavior of heterospeci c mating has been reported by many studies, such as Takafuji (1988), Fujimoto et al (1996), Takafuji et al (1997) [32][33][34], while fewer D. suzukii males showed heterospeci c mating with D. melanogaster females. Usually, reproductive interference can be regarded as a kind of "mistake" for incomplete species recognition systems. Thus, time, energy, nutrients, or gametes were wasted in heterospeci c sexual interactions, causing the tness loss of individuals involved [22]. The frequency of conspeci c courtship and mating, fecundity of females, and fertility of eggs can be in uenced by reproductive interference [22].
In addition, more potential factors resulting in population of D. suzukii decreasing by D. melanogaster were also explored. However, inconsistent with our expectations, the lifetime of D. suzukii did not become shorter when reared with D. melanogaster. Our results suggest that competition with D. melanogaster might not lead to the adults of D. suzukii having physical damage, thus affecting the longevity of adults. Therefore, the population of D. suzukii decrease was unlikely to be caused by shortening the lifetime of D. suzukii adults when reared with and competing with D. melanogaster. Besides, the larval interspeci c competition between D. suzukii and D. melanogaster also might not be a factor affecting D. suzukii population decreases, due to the mixed rearing having no signi cant effect on rates of pupation and eclosion (Table 1).

Conclusions
Generally, in this study, we found that the D. suzukii population was signi cantly decreased when rearing with D. melanogaster, which was similar to previous research. The potential factors including longevity, larval interspeci c and reproductive interference were studied. The experimental results showed that factors of longevity and larval interspeci c competition had no signi cant effect on the population of D. suzukii. However, the existence of D. melanogaster had reproductive interference effects on male D. suzukii, in which the rate of choosing conspeci c females as mating targets was signi cantly reduced, and the successful mating rate also apparently decreased. Therefore, this phenomenon can be further researched, including what features that D. melanogaster has that cause male D. suzukii not to recognize the conspeci c females. We guess Drosophila melanogaster may release some substance, such as sex hormone that distracted male D. suzukii.

Fly stocks
Adults  [35], which was changed out daily.

Longevity and fecundity
In this experiment, newly emerged (< 24 hours) individuals of the two species, Drosophila suzukii and Drosophila melanogaster, were selected. For laying eggs, one pair of D. suzukii or D. melanogaster was placed into pre-loaded transparent tubes with 20 ml solid food. Additionally, treatments with the two species mixed and reared together were also conducted. The next day, adult ies were anesthetized by CO 2 and then transferred into another transparent tube with brand new solid food until the paired ies died. The death times of adult ies in each group were recorded. All of the replaced tubes were kept in incubators set at conditions with the same light: dark photoperiod (14:10), temperature (25 ± 1 °C), and humidity (60 ± 5%) and were checked daily. The daily number of offspring was counted, and ten repetitions for each y were conducted.

Larval competition
We hypothesized that larval interspeci c competition would lead to a population change for D. suzukii. Therefore, we reared the two y species in two conditions, to con rm the larval interspeci c competition effect on adult emergence. Because interspeci c competition is a density-dependent effect on the population dynamics, when one species density increases, it causes another species density to reduce [21]. Furthermore, the species with the higher initial density is likely to exclude the species with lower initial density [36,37]. Therefore, in our experiments, we kept the same density in the two rearing conditions to make sure that the competition ability of the two ies was equal in density.
Both ies were fed on yeast water for 8 h, and then two cups containing solid food were used for egg collection. Respectively, twenty larvae of two ies were picked out into a cup with 10 g solid food for independent rearing after egg incubation. For the mixed rearing condition, ten larvae from two ies were put into the same solid food cup. All the cups were kept in incubators set at the following conditions: light: dark photoperiod (14:10), temperature (25 ± 1 °C), and humidity (60 ± 5%). Cups were checked daily, and the daily numbers of pupation and eclosion instances were counted. Respectively, ten of twenty repetitions for each y were conducted for eclosion counting and the rest were for pupation counting.
The courtship behavior observation of two ies in independent and mixed conditions In order to ensure that the ies used in behavior observations were unmated, we picked out the pupae that were going to emerge into EP tubes. All tubes were kept in incubators set at the following conditions: light: dark photoperiod (14:10), temperature (25 ± 1 °C) and humidity (60 ± 5%). The observation started 24 hours after eclosion of ies. For the independent condition, one pair of D. suzukii or D. melanogaster was put into a quartz cylinder (height: 0.5 cm, diameter: 3 cm) with a 0.5 mm quartz lid. The right amount of solid food was put in before each observation experiment, so that ies can take food in time. However, in mixed conditions, one pair of both y types was put together to make observations. We recorded a 40minute video of courtship and mating behavior with a microscope (VHX-5000, Osaka Japan, Keyence Corporation). The frequency of courtship behavior and total mating time were recorded. We performed 20 observation replicates for the independent and mixed conditions for the two y species.

Date analysis
We performed a normality test on all collected data. The longevity, offspring numbers, courtship time, and mating time data t a normal distribution, so these data were analyzed by student test (t-test). However, for the data that did not t the normal distribution, such as the frequency of correct courtship, misdirected courtship, and heterospeci c mating attempts were analyzed by the Mann-Whitney U test; the successful mating rate, pupation, and emergence rate were analyzed by the Chi-square test. All these tests were performed with R 3.6.1. and rst drafted the writing. PL and HH contributed to idea and design of the study, and comments and revisions; All authors contributed critically to the drafts. All authors read and approved the nal manuscript.